Personalized Medicine for HLA-associated Drug-hypersensitivity Reactions

Mandvi Bharadwaj; Patricia Illing; Lyudmila Kostenko


Personalized Medicine. 2010;7(5):495-516. 

In This Article

In vitro Studies of AHS Providing a Model for Allele Restricted Presentation of Drugs in DTH

We have explored allele restricted drug presentation in vitro with regards to AHS in an attempt to understand the requirements for drug presentation. In 2008, we published data showing that abacavir-treated APCs possessing HLA-B*5701, but not the related alleles HLA-B*5702, HLA-B*5703 or HLA-B*5801, can stimulate abacavir specific CD8+ T cells to produce IFN-γ and TNF-α in vitro. These alleles differ by a few residues in the F-pocket of the peptide-binding groove, a key location for peptide anchoring affecting the peptide repertoire (Figure 2). Furthermore we found that presentation was dependent on intact antigen processing pathways, leading us to propose that the immunogenic ligand is a modified self peptide, haptenated by abacavir or one of its metabolites, that is, it follows the hapten/prohapten model for presentation.[34] Since this time we have successfully eluted the antigenic ligand from abacavir-treated HLA-B*5701-positive APC and used the fractions containing identifiable peptides to selectively stimulate abacavir specific T cells [Kostenko L, Illing P. Unpublished Data]. This is the most convincing evidence yet that the immunogenic ligand in AHS is of a peptide nature. Efforts are currently underway to identify it.

Figure 2.

Antigen-presenting cells treated with abacavir and positive for HLA-B*5701, but not related alleles, can stimulate abacavir-reactive T cells.
(A) Representations of the antigen-binding cleft of HLA-B*5701 and related alleles, with differences to HLA-B*5701 highlighted by red balls and the amino acid substitutions given above each illustration. (B) An abacavir-specific T-cell line was generated in vitro as described before[34] by stimulating peripheral blood mononuclear cells from a HLA-B*5701-positive donor with abacavir. Antigen presenting cells expressing either HLA-B*5701, HLA-B*5702, HLA-B*5703 or HLA-B*5801 were then assessed for their ability to stimulate these abacavir-reactive T cells in an intracellular cytokine staining assay in the absence (upper histograms) or presence (lower histograms) of abacavir (10 µg/ml). The flow histograms show the percentage of CD3+/CD8+ cells staining positive for intracellular IFN-γ within the gated area. IFN-γ production is used as an indication of stimulation. Only abacavir-pulsed HLA-B*5701-positive antigen presenting cells are able to stimulate the abacavir-specific T cells, as demonstrated by the higher number of IFN-γ-secreting T cells in response to abacavir-treated cells in comparison to the untreated cells.

Based on these data we propose two models for the generation of HLA-B*5701 restricted immunogenicity in AHS. The first is that modification is occurring to a self-peptide that is restricted to the binding repertoire of HLA-B*5701 and that this occurs in a manner that does not alter binding but alters the face of the peptide that is exposed to the TCR. The alternative model is that haptenation of a self-peptide generates a new ligand specific for HLA-B*5701, potentially with the hapten occupying one of the key anchor sites of the HLA, and that this novel ligand is recognized by abacavir-specific T cells (Figure 3). We believe that this peptide is only able to bind HLA-B*5701; however, as single residue differences in the peptide binding groove may also alter T-cell recruitment by altering the orientation in which a peptide is displayed,[8] we also recognize that the peptide may bind related alleles but fail to stimulate T cells in this context.

Figure 3.

A model for HLA-B*5701 restricted presentation of an immunogenic ligand in abacavir hypersensitivity syndrome.
(A) Abacavir or a metabolite haptenates an unknown cellular protein, possibly alcohol dehydrogenase, which is involved in abacavir metabolism. (B) This protein then undergoes proteosome-mediated degradation to generate a peptide pool, including an abacavir-modified peptide. (C) This peptide is transferred into the ER via the TAP and loaded onto the MHC in a tapasin-dependent manner. (D) Loading is only facilitated by the specific landscape of HLA-B*5701; differences in the shape and electrostatic landscape of the binding grooves of other alleles prevent the loading and binding of this peptide.
ER: Endoplasmic reticulum; TAP: Transporter associated with antigen processing.

It is now a priority to identify the peptide ligand, not only to clarify these issues, but to identify the culprit functional groups of abacavir responsible for its generation, potentially providing a starting point for drug redesign to avoid hypersensitivity in HLA-B*5701 positive individuals. Furthermore, understanding what is occurring in AHS may help inform the study of what is occurring in some of the other HLA-associated hypersensitivities.

By contrast to the situation in AHS, in vitro studies with carbamazepine, indicate that drug presentation is independent of antigen processing, and is contingent on continual drug presence, suggesting binding may occur via a labile interaction with the HLA (the p-i concept).[48–50] In such instances it is possible that the associated HLA, in complex with one of its peptide repertoire, generates a unique structure that is able to interact with the drug, or drug metabolite, with sufficient stability to stimulate a T cell via its TCR, whilst other alleles do not have the correct underlying structure and/or peptide repertoire. To understand this reaction, it is necessary to study the specific requirements for stimulating carbamazepine-specific T cells from HLA-B*1502 positive individuals who have experienced carbamazepine–SJS/TEN.